Vascular smooth muscle cell (VSMC) migration is critically important in neointimal formation following vascular injury and atherosclerotic lesion formation. During the last grant period, we showed that VSMC migration in response to platelet-derived growth factor (PDGF) is mediated by reactive oxygen species (ROS) derived from the Nox1 NADPH oxidase via regulation of cofilin, an actin binding and severing protein. We also found that the Nox4 NADPH oxidase and its regulator Poldip2 are required for migration via regulation of the small molecular weight G-protein Rho and focal adhesion turnover. In this grant period, we propose to extend our investigation of the signaling pathways by which these two NADPH oxidases separately and specifically regulate the reorganization of the cytoskeleton that is required for VSMC migration. We propose that the subcellular location of ROS production dictates the cellular response. Thus, production of ROS by PDGF-induced activation of Nox1 in lamellipodia regulates acute actin polymerization/depolymerization during the cytoskeletal reorganization that accompanies lamellipodial protrusion, while the production of H2O2 by integrin-mediated Nox4 activation in focal adhesions regulates focal adhesion turnover and stabilizes actin filaments via oxidation. To test this hypothesis, three specific aims will be accomplished. First, we will define the signaling pathways by which Nox1 regulates PDGF- induced lamellipodial formation in VSMCs. We will investigate factors controlling actin polymerization in the lamellipodia, including coronin 1b, Arp2/3 and slingshot 1L phosphatase (SSH1L), and their relationship to Nox1. Second, we plan to determine the role of Nox4 in integrin-mediated actin oxidation and focal adhesion turnover during migration. We will test the hypothesis that Nox4 critically regulates focal adhesion turnover and migration by virtue of its ability to oxidize actin, thus leading to changes in the binding of actin regulatory proteins, and to facilitate RhoA-mediated focal adhesion formation. Finally, we will test this model in vivo by determining the role of Nox4/Poldip2-derived H2O2 production in neointimal formation. We plan to use genetrap mice in which Poldip2 expression is reduced, as well as Nox4 knockout mice, to investigate the role of Nox4/Poldip2 and their targets using an in vivo model of wire injury-induced neointimal formation that is dependent upon VSMC migration. Together, these aims will provide new insight into how NADPH oxidases mediate VSMC migration and therefore lesion formation. Such information may lead to the development of new therapeutic strategies that can be carefully and specifically targeted to the critically important events in disease initiation. PUBLIC HEALTH RELEVANCE: This proposal will investigate the roles of the vascular smooth muscle cell (VSMC) NADPH oxidases, Nox1 and Nox4, in PDGF-induced migration, and the molecular mechanisms by which reactive oxygen species derived from these enzymes mediate the early cellular events that lead to VSMC migration. VSMC migration is clinically important during neointimal hyperplasia after vascular injury and atherosclerotic lesion formation, but despite advances in the design of the mechanical devices, and despite new adjunctive medical therapies, complications from atherosclerosis and restenosis account for >36% of all deaths (per the AHA website). Understanding the mechanisms underlying migration may lead to the development of new therapeutic strategies that can be carefully and specifically targeted to the critically important events in disease initiation.